Suppressing ROS generation by apocynin inhibited cyclic stretch-induced inflammatory reaction in HPDLCs via a caspase-1 dependent pathway
Introduction
Periodontal ligament (PDL) is the primary target of mechanical forces aroused during occlusion, mastication and orthodontic treatment [1], [2], [3]. Human periodontal ligament cells (HPDLCs) are the sensors of such mechanical stimuli and respond to them through activated metabolism, proliferation, osteoblastic differentiation and inflammation, thus play an important role in biological processes of the maintenance of periodontal tissue homeostasis, repair, and remodelling [1], [2]. It has been reported that HPDLCs also participated in some pathological process such as mechanical force-induced periodontitis, which was aroused by abnormal mechanical stimulations from occlusal overloading or improper orthodontic treatment [4], [5], [6], [7], [8], [9].
Our recent study showed that cyclic stretch activated Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and induced inflammatory reaction including the secretion of pro-inflammation cytokines and pyroptosis in HPDLCs [10], and confirmed the role of NLRP3 inflammasome in the mechanical force-induced periodontal inflammatory reaction. NLRP3 inflammasome is a high-molecular-weight oligomer formed by oligomerization of NLRP3, ASC and caspase-1 [11], and has been reported to play a critical role in the development of periodontitis [12]. In the oligomer, pro-caspase-1 is converted to its active form, caspase-1, which will subsequently cleave its substrates such as pro-interleukin-1β (pro-IL-1β) and pro-interleukin-18 (pro-IL-18) to their bioactive forms (IL-1β and IL-18) [13].
Several cellular events, such as mitochondrial dysfunction, reactive oxygen species (ROS) production, potassium efflux, and cell swelling, have been proposed as upstream signals for the activation of NLRP3 inflammasome [14], [15], [16]. It has been reported that lipopolysaccharide (LPS) and advanced products of glycation end activated NLRP3 inflammasome and induced inflammatory reaction in HPDLCs through production of ROS [11], [17]. These findings confirmed the involvement of ROS in the pathogen-related activation of inflammasome and the pathogen-induced periodontal inflammatory reaction. Whether ROS are involved in force-related activation of inflammasomes and force-induced periodontal inflammatory reaction still remains unclear. Interestingly, a recent study confirmed the activation of NLRP3 inflammasome in mechanical stretch-induced lung injury via ROS production [18]. Furthermore, another recent study showed that ROS level in capillary blood increased after short-term (24 h) orthodontic force loading in patients treated with fixed orthodontic appliances [19]. Therefore, it’s reasonable to speculate that ROS might also play a role in force-related activation of inflammasomes and force-induced periodontal inflammatory reaction.
ROS are considered to be a double-edged sword in periodontal diseases [20]. At low concentrations, ROS stimulate the proliferation and differentiation of HPDLCs, while at higher concentrations, they may have cytotoxic effects on periodontal tissues [21], [22], [23], [24], [25]. Oxidative stress or ROS accumulation is a significant process in the pathogenesis of several oral diseases [26], [27].
Previous studies have found that ROS are produced in the mitochondrial respiratory chain during cellular respiration and oxidative reactions which are catalyzed by NADPH oxidase, xanthine oxidase or Lamino-acid-oxidase [28]. The key producer of ROS in many cells is NADPH oxidase, comprising membrane and cytosolic components, which actively communicates during the host responses to a wide variety of stimuli [29]. NADPH Oxidase is a multi-subunit complex composed of six subunits [29]. Apocynin (4′-hydroxy-3′-methoxyacetophenone or acetovanillone) can block the activity of NADPH oxidase by preventing the phosphorylation of the p47phox subunit of NADPH Oxidase [30]. Many studies have shown the anti-inflammatory and antioxidation effects of apocynin in various human diseases, including LPS induced periodontal diseases [11]. However, the role of ROS in cyclic stretch-induced inflammatory reaction in HPDLCs still remained to be elucidated.
Taken together, we hypothesize that cyclic stretch induces inflammatory reaction in HPDLCs via a ROS-related signaling pathway and apocynin as ROS inhibitor can help suppress this cyclic stretch-induced inflammatory reaction. The findings of this study may help us better understand the mechanism of the mechanical force-related periodontal inflammation and provide evidences to support antioxidants as a hopeful treatment for these kinds of periodontal diseases.
Section snippets
Cell cultures and cyclic stretch loading
HPDLCs were cultured following the method reported by us previously, which has been described by Zhuang et al. [31] in detail. In brief, pieces of PDL tissues were harvested from the middle of the root surface of extracted premolars of teenagers (11–16 years old) for orthodontic reason with the informed consents from their parents. Collected PDL tissues were attached to a cell culture dish and incubated with high-glucose Dulbecco’s modification of Eagle’s medium (DMEM, HyClone, Logan, UT, USA)
Cyclic stretch induced the generation of ROS in HPDLCs
Result of cellular ROS detection by Flow Cytometry showed that the expression of ROS in HPDLCs increased in response to 3 h and 6 h cyclic stretch (P < 0.01 VS control for stretched-3 h group, P < 0.001 VS control for stretched-6 h group) and it went down after 12 h and 24 h cyclic stretching (P < 0.001 VS stretched-6 h group) (Fig. 1).
Cyclic stretch induced pyroptosis in HPDLCs
Flow cytometric analysis demonstrated that the pyroptotic rate of HPDLCs in response to 6 h cyclic stretch increased significantly, compared with non-stretched
Discussion
The PDL functions as a cushion to withstand mechanical forces applied to teeth. PDLCs are stimulated by forces applied during mastication, occlusal contact, and orthodontic treatment and produce local factors that participate not only in the maintenance and remodeling of the ligament itself, but also in the metabolism of adjacent alveolar bone [35]. Physiological mechanical forces help to maintain the balance in periodontal tissue, while abnormal mechanical forces such as traumatic occlusion
Conclusions
The present study showed for the first time that the cyclic stretch induced inflammatory reaction in HPDLCs through stimulating the production of ROS, probably via a caspase-1-dependent mechanism. Apocynin could be a promising option for protecting periodontal tissue against force-related inflammation. Nevertheless, the exact regulating mechanisms in this force-related ROS/NLRP3/caspase-1 signaling pathway need to be further elucidated.
Funding
This work was supported by National Natural Science Foundation of China (grant numbers 31470903, 31270991, 30900282), Shanghai Municipal Health Commission (grant number 201940009), Shanghai Summit & Plateau Disciplines, Shanghai Pujiang Program (grant number 13PJD021), and Science and Technology Commission of Shanghai (grant number 10QA1404200, 08411961500, 07ZR14070).
Compliance with Ethical Standards
The experimental protocol was reviewed and approved by the Ethics Committee of Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine. The relevant judgment’s reference number is [2017]96.
CRediT authorship contribution statement
Yingying Wang: Conceptualization, Data curation, Investigation, Methodology, Software, Writing - original draft, Writing - review & editing. Yingshuang Song: Data curation, Visualization. Qi Zhong: Data curation, Software. Yaqin Wu: Conceptualization, Methodology. Jiabao Zhuang: Methodology, Supervision. Fang Qu: Conceptualization, Supervision. Chun Xu: Conceptualization, Funding acquisition, Resources, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Yingying Wang and Yingshuang Song should be considered joint first author.